isolation of microtubules and microtubule proteinsbiochem.du.ac.in/web/uploads/10 isolation...

UNIT 3.29Isolation of Microtubules andMicrotubule Proteins

J. Avila,1 H. Soares,2 M.L. Fanarraga,3 and J.C. Zabala3

1Centro de Biologıa Molecular (CSIC). Facultad de Ciencias, Universidad Autonoma deMadrid, Cantoblanco, Madrid, Spain2Instituto Gulbenkian de Ciencia, 2781-901, Oeiras, and Escola Superior de Tecnologia daSaude de Lisboa, Lisboa, Portugal3Departamento de Biologıa Molecular, Facultad de Medicina, Universidad de Cantabria-IFIMAV, Santander, Spain

ABSTRACT

This unit describes various protocols for the isolation and purification of the main con-stituents of microtubules, chiefly α- and β-tubulin, and the most significant microtubuleassociated proteins (MAPs), specifically MAP1A, MAP1B, MAP2, and tau. We includea classical isolation method for soluble tubulin heterodimer as the first basic purificationprotocol. In addition, we show how to analyze the tubulin and MAPs obtained after aphosphocellulose chromatography purification procedure. This unit also details a pow-erful and simple method to determine the native state of the purified tubulin based onone-dimensional electrophoresis under nondenaturing conditions (UNIT 6.5). The last pro-tocol describes the application of a new technique that allows visualizing the quality ofpolymerized microtubules based on atomic force microscopy (AFM). Curr. Protoc. CellBiol. 39:3.29.1-3.29.28. C© 2008 by John Wiley & Sons, Inc.

Keywords: tubulin � microtubule � MAP1A � MAP1B � MAP2 � tau �

native gel electrophoresis � atomic force microscopy

INTRODUCTION

The different morphologies displayed by cells from different tissues—for example thegreat diversity of neurons—is the result of the assembly of different cell scaffolds.

There are three different components in the cell scaffold known as the cytoskeleton. Theseare microtubules (Fig. 3.29.1), microfilaments, and intermediate filaments. Microtubulesare particularly abundant in brain tissue and their components represent ∼20% of thetotal protein content from soluble brain extract, with α- and β-tubulin heterodimers themajor components.

Microtubules are tubular cytoplasmic fibers with a diameter of 24 nm (Fig. 3.29.2).In addition to their role in the determination of cell shape they also play fundamentalroles in other common cellular functions, such as intracellular transport, chromosomesegregation (a specific form of transport), and flagella/cilia-mediated cell motility. Inneurons, besides their function in determining neuron shape, microtubules seem to playessential roles in the formation and maturation of the axon and dendrites. The majority,if not all, of the microtubule functions are based on the capacity of these polymers topolymerize/depolymerize. The capacity of microtubules for assembly-disassembly wasmimicked in vitro in the pioneer work of Weisenberg (1972), who reproduced condi-tions for the assembly of these polymers using a brain cell extract. In this work it wasestablished that microtubule polymerization depends on the amount of tubulin and that,at least in vitro, a critical concentration of this protein is needed for polymerization intomicrotubules. Since there is a large amount of tubulin in a brain cell extract, it is possible

Current Protocols in Cell Biology 3.29.1-3.29.28, June 2008Published online June 2008 in Wiley Interscience (www.interscience.wiley.com).DOI: 10.1002/0471143030.cb0329s39Copyright C© 2008 John Wiley & Sons, Inc.

SubcellularFractionationand Isolation ofOrganelles

3.29.1

Supplement 39

Isolation ofMicrotubules and

MicrotubuleProteins

3.29.2

Supplement 39 Current Protocols in Cell Biology

Figure 3.29.1 Microtubule networks of cerebellar cultured granule cells, as visualized by im-munofluorescence microscopy using a specific antibody against the α-tubulin subunit (DM1A).

Figure 3.29.2 Electron micrograph of brain microtubules assembled after a single polymer-ization cycle. Scale bar represents 1 µm. For color version of this figure see http://www.currentprotocols.com.

to induce microtubule assembly in vitro. In addition, Weisenberg also found that thepresence of GTP and the absence of calcium were both needed to properly carry outmicrotubule assembly. These observations served as the basis of various methods for mi-crotubule protein purification (Borisy and Olmsted, 1972; Shelanski et al., 1973). At thattime it was also found that microtubules are induced to assemble at warm temperatures.In contrast to tubulins from Antarctic fishes that polymerize at low temperatures, mam-malian microtubules are not cold-stable and depolymerize very fast at 4◦C. The observedtemperature dependence seems to reside in differences in the polymerization energeticsand kinetics of specific tubulin polypeptides. Thus, warming a cytosolic brain extractpromotes microtubule polymerization, and the microtubules can be easily collected bycentrifugation. But, because in vitro–polymerized mammalian microtubules depolymer-ize in the cold, after centrifugation, collected microtubules can be depolymerized using acold buffer. After depolymerization, the solution can again be centrifuged to remove con-taminants and those microtubule fragments that are not competent for depolymerization.


3.29.3

Current Protocols in Cell Biology Supplement 39

This sequence of warm-polymerization and cold-depolymerization can be repeated twoor more times until the desired degree of purity of the microtubules is achieved.

The quality and quantity of tubulins and proteins associated with microtubules (MAPs)obtained can be followed by SDS-PAGE analysis (UNIT 6.1). From this analysis it ispossible to observe the main microtubule components—two polypeptides or subunits,α- and β-tubulin (for reviews see: Vallee et al., 1986; Matus, 1988; Avila, 1990). MAPsare classified in three major families. The first protein family comprises the MAP1polypeptides: MAP1A, MAP1B, and MAP1C (or dynein, a motor protein). The secondfamily is composed of four structurally related proteins derived from a single gene:MAP2A, MAP2B, MAP2C, and MAP2D. The third family, present in a lower amount,is composed of several related polypeptides (tau proteins), or isoforms expressed from aunique gene. All these MAPs preferentially bind to the C-terminus of tubulins (Serranoet al., 1984a,b, 1986).

In this unit, we describe protocols for the purification of tubulins (Basic Protocols 1 and3 and Alternate Protocol 2), MAP1 (Basic Protocol 4), MAP2 (Basic Protocol 5), and tauproteins (Basic Protocol 6), but not for other MAPs or motor proteins (such as dynein). Wealso describe how to assemble microtubules in vitro in the presence of paclitaxel (Taxol;Basic Protocol 2 and Alternate Protocol 1) and protocols for native gel electrophoresis(Support Protocol 1) and atomic force microscopy (AFM; Support Protocol 2).

BASICPROTOCOL 1

ISOLATION OF MICROTUBULE PROTEIN BY CYCLES OFPOLYMERIZATION AND DEPOLYMERIZATION

Many similar microtubule protein purification methods have been developed, published,and used for the purification of microtubule proteins (Shelanski et al., 1973; Williams andLee, 1982; Vallee, 1986a). Most of these are based on the reversible assembly capacity ofmicrotubule proteins in the presence or in the absence of assembly-promoting agents, suchas glycerol or dimethyl sulfoxide (DMSO). Here we describe the isolation of brain mi-crotubule proteins by temperature-dependent cycles of polymerization-depolymerizationusing glycerol and DMSO in the first and second polymerization cycles, respectively(Fig. 3.29.3). Figure 3.29.3 illustrates the results of the step-by-step protocol that wefollow in our laboratories. This method can be applied to brain tissues from differentsources, i.e., porcine, bovine, rat, etc. The protocol is appropriate for a medium-scalepreparation (1 to 3 cow or pig brains).

Materials

One to three brains (100 to 500 g total start-up tissue; the weight of a pig brain is∼100 g)

Phosphate-buffered saline, pH 6.7 (PBS; APPENDIX 2A), 4◦CIsotonic buffer (see recipe), 4◦CProtease inhibitors (see recipe)Buffer A (see recipe), 4◦CGlycerolPMSFGTP (see recipe)Dimethyl sulfoxide (DMSO)

Plastic wrapIce-water bathNo.10 scalpel blade500-ml beakerPotter homogenizer (Teflon-in-glass homogenizer), as large as possibleCentrifuge (if possible two centrifuges, one at 4◦ to 6◦C and the other at 25◦ to

30◦C): Sorvall RC-5B or equivalent Beckman J2, J21 series, or Avanti J-25


MicrotubuleProteins

3.29.4


Figure 3.29.3 Characterization of pelleted protein after one (A), and three polymerization cycles.(B) In (A), the presence of MAP1, MAP2, neurofilament proteins (NFH, NFM and NFL), tau protein,tubulin subunits, and actin are indicated. The gel was stained with Coomassie blue as explainedin UNIT 6.6.

Centrifuge rotors: GSA or JA-10; type 50.2Ti and 70.1 Ti for the BeckmanGraduated flask26.3-ml ultracentrifuge tubes35◦C rocking incubatorUltracentrifuge Beckman L8-70, Optima XL-100 or 120

Additional reagents and equipment for measuring protein concentration(APPENDIX 3B)

Obtain the brains1. Make an appointment with veterinarians at the slaughterhouse and request fresh

brains to be picked up immediately after animal slaughter.

2. Wrap brains carefully with plastic before introducing in the ice-water bath. Placebrains on ice during transport to the laboratory (<1 hr in our case). After arriving atthe laboratory start the preparation of the cytosolic extracts (immediately).

Prepare brains3. Rinse brains well, three times, each time with 100 ml cold PBS buffer and keep them

cold throughout the following steps until the first polymerization cycle.

4. Remove the meninges, the blood clots, and most of the peripheral blood vessels fromthe brain.

5. Chop the brains into smaller pieces (1- to 2-cm) using a no. 10 scalpel blade.

6. Weigh the tissue in a tared 500-ml cold beaker.

Homogenize the brain tissue7. Add 0.75 to 1 ml of isotonic buffer per gram of tissue.

The use of the isotonic buffer for tissue homogenization facilitates the removal of neuro-filaments in the following microtubule isolation process.


3.29.5


8. Add PMSF to a final concentration of 1 mM. Add aprotinin, leupeptin, and pepstatinto a final concentration of 1 µM each (see recipe for protease inhibitors in Reagentsand Solutions).

9. Disrupt brain tissues at 4◦C by using a Potter homogenizer, which allows a fast andreproducible homogenization.

Use the largest Potter you have and homogenize as many times as required depending onthe total volume (see Critical Parameters).

10. Clear the crude brain homogenate by centrifuging 30 min at 25,000 × g, 4◦C.

If necessary, since the volume is too high, a rotor type GSA (Dupont-Sorvall) or equivalentJA-10 in a Beckman J2-21, or Avanti J-25 centrifuge can be used.

11. Decant the supernatant in a cold 500-ml beaker and, if necessary for further clarifi-cation, centrifuge again for 30 to 45 min at 25,000 × g, 4◦C.

12. Discard the pellet and take the supernatant. Take a small aliquot for analysis inSDS-PAGE (UNIT 6.1), native electrophoresis (Support Protocol 1), and for proteindetermination (APPENDIX 3B).

From an initial 100 g we usually get 150 ml at 40 mg/ml at this point, and, of this amount,∼20% represents microtubule proteins.

13. Measure the total volume of the cytosolic extract in a graduated flask and make it 1×buffer A by adding for each 90 ml of extract, 10 ml of 10× buffer A. Add glycerolto a final concentration of 25%(v/v) to 30%(v/v) (Krauhs et al., 1981).

Be aware that some commercial glycerol preparations have a concentration of 87%.

14. Add PMSF to a final concentration of 1 mM to prevent protein degradation bynonspecific proteolysis. Finally, add 1 M GTP (1000×) to a final concentration of1 mM.

Perform first polymerization15. Transfer the solution to 26.3-ml ultracentrifuge tubes and balance them to ±0.01 g.

Incubate solution for 30 min at 35◦C with gentle agitation.

16. Ultracentrifuge the mixture 45 min at 100,000 × g, 25◦C, using a prewarmed (30◦C)rotor.

We use the 50.2 Ti rotor in a Beckman centrifuge.

17. Discard the supernatant and take the pellet, which should be translucent.

If necessary, pellets can be frozen at −80◦C at this point without much loss of function ofthe polymerized tubulin. We usually get 300 to 400 mg protein at this point, determinedby Lowry’s method.

Perform first depolymerization18. Resuspend the pellet in cold buffer A at 10 to 20 mg/ml. Let the mixture to stand in

an ice-water bath for 30 min.

Intermittent homogenization of the mixture with a Teflon-in-glass homogenizer (10 strokes)will facilitate microtubule depolymerization.

19. Centrifuge 15 to 20 min at 25,000 × g, 4◦C using a precooled rotor Beckman 50.2Ti. Discard the pellet and keep the supernatant.

The pellet at this point is enriched in the so-called cold-resistant microtubule fractionand denatured protein. It is not advisable to centrifuge at very high speed, to avoid lossof “competent” microtubule proteins.


MicrotubuleProteins

3.29.6


20. Add PMSF and GTP to the supernatant to a final concentration of 1 mM (add 1/1000volume of 1 M GTP). Add DMSO to a final 10% concentration.

CAUTION: DMSO is hazardous: see APPENDIX 2A for guidelines on handling, storage,and disposal.

Perform second polymerization21. Transfer the solution to 26.3-ml ultracentrifuge tubes and balance them to within

±0.01 g. Incubate the tubes 30 min at 35◦C with gentle agitation.

22. Collect the microtubules polymerized in the second cycle by ultracentrifuging 40 minat 180,000 × g (Beckman 70.1 Ti at 50,000 rpm), 25◦ to 30◦C.

If at this point there is only one rotor available, wash the rotor with hot water to rapidlyreach the appropriate temperature.

23. Discard the supernatant. To finish the second cycle resuspend the pellet in 6 ml ofcold 0.5× buffer A. Let the mixture stand in an ice-water bath for 30 min.

The pelleted protein can be frozen in liquid nitrogen or at −80◦C for further use. Weusually get 60 to 80 mg protein at this point.

Occasional homogenization of the mixture with a Teflon-in-glass homogenizer (10 strokes)will facilitate depolymerization of microtubules.

24. Centrifuge 20 min at 25,000 × g, 4◦C, using a precooled rotor. Discard the pellet.Measure protein concentration in the supernatant by using a spectrophotometerabsorbance at 280 nm and 260 nm. (APPENDIX 3B)

The extinction coefficient of tubulin at 280 nm is 115,000 M−1cm−1 [1.15 (mg/ml)−1

cm−1]. This extinction coefficient is calculated from tubulin sequences and includes thecontribution of the two bound guanine nucleotides to the absorbance at 280 nm. For thisreason, we use the formula A280/1.15 = mg/ml of the sample. Also, this formula mightbe improved if we take into account the contribution of nucleotides to the absorbance at260 nm: 1.45 × A280 − 0.74 × A260 = mg/ml of tubulin. The isolated tubulin competentto polymerize can be stored at −80◦C for several months or until further steps.

BASICPROTOCOL 2

ISOLATION OF MICROTUBULE PROTEIN USING PACLITAXEL (TAXOL)

An alternative single-step method to isolate polymerized microtubule proteins is based onthe fact that microtubules assemble and remain stable in the presence of paclitaxel (Shiffet al., 1979). This allows one to sediment them and to resuspend them in a cold bufferwithout depolymerization (Borisy and Olmsted, 1972). Moreover, an advantage of thisprocedure is the fact that MAPs can be easily dissociated from these stable microtubulesby exposing them to an elevated ionic strength. Additionally, the use of paclitaxel permitsthe purification of tubulin from sources where the amount of this protein is low.

Materials

Bovine or porcine brain tissue (10 g)Phosphate-buffered saline (APPENDIX 2A)Buffer A (see recipe)Protease inhibitors (see recipe)Paclitaxel (Taxol; see recipe)GTP (see recipe)PMSFSucrose underlayer solution (1 ml per centrifuge tube containing 10% sucrose,

10 µM paclitaxel, and 0.5 mM GTP)

Scalpel blade, No.10500-ml beaker


3.29.7


Potter homogenizer (Teflon-in-glass homogenizer)Centrifuge rotors: GSA or JA-10. Type 50.2Ti and 70.1 Ti for the BeckmanUltracentrifuge Beckman L8-70, Optima XL-100 or 12013.5-ml ultracentrifuge tubes30◦C rocking incubatorPasteur pipet

Prepare cytosolic extract1. Obtain fresh brains from the slaughterhouse as described in Basic Protocol 1.

2. Place and keep brains on ice until arrival at the laboratory, as described in BasicProtocol 1.

Although we use fragments of any region of porcine brain, whole brains from other sourcescan also be used.

3. Rinse brains well, three times, each time with 100 ml cold PBS buffer and keep themcold throughout the following steps until the first polymerization cycle.

4. Remove the meninges, the blood clots, and most of the peripheral blood vessels fromthe brain.

5. Chop the brains into smaller pieces (1 to 2 cm) using a scalpel blade, no. 10. Weighthe tissue in a tared 500-ml cold beaker.

6. Add 15 ml buffer A (1.5 ml per g of tissue).

7. Disrupt brain tissues at 4◦C by using a Potter homogenizer.

Follow recommendations in Basic Protocol 1.

8. Add freshly prepared PMSF to a final concentration of 1 mM in DMSO. Also add thefollowing protease inhibitors to a final concentration of 1 µM: aprotinin, leupeptinand pepstatin (see recipe for protease inhibitors in Reagents and Solutions).

9. Clear the crude brain homogenate by ultracentrifuging 40 min at 100,000 × g, 4◦C.Use a precooled rotor (Beckman 50.2 Ti or similar).

Polymerize microtubules10. Discard the pellet and add paclitaxel to the supernatant to a final concentration of

20 µM, GTP to a final concentration of 1 mM, and PMSF to a final concentration of1 mM.

11. Transfer the solution to 13.5-ml ultracentrifuge tubes and balance them to within±0.01 g. Incubate at 30 min at 30◦C with gentle agitation.

12. With a Pasteur pipet introduce 1 ml of a 30◦C prewarmed sucrose solution verygently at the bottom of the tube.

13. Collect the assembled microtubules in the polymerization cycle by ultracentrifuging30 min at 180,000 × g (prewarmed rotor Beckman 70.1 Ti), 25◦ to 30◦C.

14. Discard the supernatant and keep the translucent pellet. Resuspend pellet in 3 mlbuffer A containing 20 µM paclitaxel and 1 mM GTP.

These microtubule preparations, containing assembled microtubules, can be used asa source for isolating MAPs, such as MAP1B (see below) or they can be stored in therefrigerator (4◦C). You must be aware that storage for >1 week will enrich the preparationin unpolymerized tubulin. To store samples for longer periods keep the preparation at−80◦C.


MicrotubuleProteins

3.29.8


ALTERNATEPROTOCOL 1

MICROTUBULE POLYMERIZATION IN THE PRESENCE OF PACLITAXEL

Microtubule ??proteins (tubulin and MAPs) are prepared by repeated cycles of assembly/disassembly using a temperature-dependent procedure as described in Basic Protocol 1.Purified microtubule heterodimers are incubated 30 to 40 min with 20 to 25 µM paclitaxel(taxol) at 35◦C in BRB80 buffer (see recipe) containing 1 mM GTP and 30% glycerol.The pellet corresponding to the polymerized microtubules is obtained by centrifuging30 min at 45,000 × g, 30◦C through a 50% sucrose cushion containing 1 mM GTP.

BASICPROTOCOL 3

ISOLATION OF TUBULIN DIMERS FROM MICROTUBULE PROTEINS

Tubulin contains very acidic regions, mainly in its C-terminal domain (Krauhs et al.,1981), which allows its fractionation from many other proteins by ion-exchangechromatography (anionic or cationic). Two types of resins have been successfully used topurify tubulin: cationic resins such as DEAE-cellulose (or DEAE-Sephadex) and anionicresins like phosphocellulose. However, it is possible to use any other type of ion-exchangeresins such as Mono-Q or Mono-S from Pharmacia, or HiTrap columns. Nevertheless, allthese chromatographic columns show advantages and disadvantages. For example, whilethe use of DEAE-cellulose is a slow procedure, the use of phosphocellulose columnsmight result in a partial inactivation of tubulin due to magnesium ion chelation (Williamsand Detrich, 1979). Likewise, highly purified tubulin can be obtained from cationiccolumns, but the fact that tubulin binds to these resins requires the use of a salt gradientto elute the protein, which might result in tubulin denaturation. Therefore, tubulin elutionfrom these columns requires a further step of salt removal by dialysis or ultrafiltration. Inthis protocol we describe the classical procedure to purify tubulin using phosphocellulosechromatography. This method has been chosen because it is the fastest and does notneed the additional dialysis step that could also result in a partial denaturation of tubulin.

Materials

Resin: Whatman P11 Cellulose Phosphate0.5 M NaOH0.5 M HClBuffer A (see recipe)50 mg of porcine brain microtubules stored at −80◦C or in liquid nitrogen (Basic

Protocol 1)

Buchner funnelXK50/20 (Length 20 cm, i.d. 50 mm; Pharmacia) column including:

Thermostat jacketFlow adaptorFlanged tubing at both ends for direct connection to valvesPumpsUV monitors if using an FPLC or an AKTA system

Teflon-in-glass homogenizerIce-water bathUltracentrifuge

Additional reagents and equipment for isolating microtubule proteins (BasicProtocol 1), determining protein concentration (APPENDIX 3B), SDS-PAGE(UNIT 6.1), and nondenaturing gel electrophoresis (Support Protocol 1)

Precycle the ion exchanger1. Take 100 g of resin and stir into 1.5 liter of 0.5 M NaOH for 30 min at room

temperature. Filter the resin in a Buchner funnel and wash with water several times.

Alternatively, stir the resin in a beaker and let it settle by gravity for a few minutes anddecant the supernatant.


3.29.9


2. After the wash, treat the resin with 1.5 liter of 0.5 M HCl as described above. Filteror decant and wash several times with water.

3. Repeat this procedure until a neutral pH is attained.

4. Next, filter and stir the resin in 0.5× buffer A.

5. Add the resin to an XK 50 column (∼150 ml) and equilibrate with 0.5× buffer Auntil the absorbance at 280 nm returns to baseline.

Prepare sample6. Collect microtubule proteins (10 mg/ml), isolated following Basic Protocol 1, by

depolymerization of microtubules preparing a homogenate in cold 0.5× buffer Awith the help of a Teflon-in-glass homogenizer.

7. Leave homogenized sample in ice-water for 30 min.


8. Ultracentrifuge the homogenate 15 min at 25,000 × g, 4◦C, using a precooled rotor.Discard the pellet and keep the supernatant.

We currently use 50.2 Ti or 70.1 Ti rotors in a Beckman centrifuge in our laboratory.

9. Adjust the final protein concentration of the supernatant to ∼10 mg/ml and loadit onto a prepacked XK 50 phosphocellulose column (1 ml column/mg protein)equilibrated in 0.5× buffer A.

The first time you use the resin you must take into account that you will probably lose sometubulin. This is due to the fact that some will irreversibly bind to the phosphocellulose.This could be overcome or minimized by previously loading in the column with 1 g ofBSA diluted in the same buffer, followed by several washes with the salt-containing bufferbefore re-equilibration.

Figure 3.29.4 (A) Chromatographic elution profile obtained at 280 nm of absorbance from a gelfiltration analysis of phosphocellulose-purified tubulin chromatographed through a Sepharose 4Bcolumn. (B) Denaturing SDS gel (8.5% polyacrylamide) analysis of the peak corresponding to amolecular mass of 110 kDa eluted from the column. The gel was stained with Coomassie blue asexplained in UNIT 6.6. Abbreviations: Vo, void volume.


MicrotubuleProteins

3.29.10


10. Collect 0.5-ml fractions from the column.

Tubulin, which will be present in the first fractions after the void volume of the column,can be detected by measuring absorbance at 280 nm (Fig. 3.29.4).

11. Determine protein concentration by Bradford, Lowry, or BCA, and measure theabsorbance at 280/260 as described in APPENDIX 3B.

12. To finish, analyze the quality of purified tubulin by SDS-PAGE (UNIT 6.1) and bynondenaturing gel electrophoresis (Support Protocol 1).

13. Add to the tubulin-containing fractions the amount of 10× buffer A to obtain 1×buffer A, and GTP to a final concentration of 1 mM. Freeze fractions immediatelyin liquid nitrogen or at −80◦C.

Other microtubule associated proteins (MAPs) can be eluted from the column using bufferA containing 2 M NaCl (Basic Protocol 2 and Alternate Protocols 4 and 5).

BASICPROTOCOL 4

ISOLATION OF MAP1A/1B

Microtubule-associated proteins comprise a heterogeneous group of polypeptides whichdisplay different structural characteristics, are differentially expressed during develop-ment, and are localized at different sites in neurons. A good example of these differencescan be found between proteins termed MAP1A and MAP1B (Bloom et al., 1984; 1985).MAP1B is localized in the developing rat brain (Tucker et al., 1989), decreasing its expres-sion dramatically at the end of the neuronal differentiation. On the other hand, MAP1Ais expressed at later developmental stages and its level of expression remains constantthrough adulthood. MAP1B and the protein tau are mostly axonal proteins, whereas thehigh-molecular-weight forms of MAP2 are mostly found in dendrites (Matus, 1988).

Different MAPs display different characteristics which have served in developing di-verse strategies and schemes to purify them. For instance, MAP2 and tau proteins areheat-resistant proteins, whereas MAP1A/1B denature at high temperature. Yet, sinceMAP1A/1B preferentially bind to a cluster of acidic amino acids present at the carboxy-terminus of tubulin (Tucker et al., 1989; Cross et al., 1991), molecules like poly-L-asparticacid can be employed to compete with the interaction of these MAPs with tubulin (Fujiiet al., 1990). Moreover, MAP1B can be isolated from the brain of newborn rats, whereasthe most appropriate source for other MAPs is the adult brain. Similarly, the differentialproportion of axons/somatodendritic tissues between white and gray matters can also beused to differentially purify MAP1 (white matter) and MAP2 (gray matter), minimizingcontaminations (Vallee, 1986b), while either of the two matters can be used to purifyMAP1A. In the case of tau, the expression pattern of the different isoforms is clearlydifferent between white and gray matter, and there is a dramatic decrease in the amountof two tau isoforms in gray matter (de Ancos and Avila, 1993).

MAP1B/1A are both thermolabile proteins and cannot be isolated through a heat-treatment step. Instead, the addition of poly-L-aspartic to taxol-stabilized microtubules de-taches MAP1A/1B proteins from these polymers. This step is performed in a PIPES bufferwhere the interaction of MAP1 proteins with tubulin is weakened (Pedrotti et al., 1993).MAP1 proteins are next fractionated from taxol-polymerized microtubules by centrifuga-tion, being further purified using an FPLC (fast protein liquid chromatography) or AKTAsystem (Pazzagli and Avila, 1994). This particular protocol uses adult rat or porcinebrains as the source of MAP1A. MAP1B is isolated from whole newborn-rat brains.These sources were chosen taking into consideration the information described above.

Materials

White matter from a cow or ten brains from adults rats (MAP1A)Twenty brains from newborns rats (MAP1B)


3.29.11


Buffer B (see recipe)GTPPaclitaxel (Taxol; see recipe)Poly-L-asparticBuffer C (see recipe)Buffer D (see recipe)Buffer A (see recipe)

10.4-ml centrifuge tubesIce-water bathCentrifugeCentrifuge rotors: GSA or JA-10. Type 50.2Ti and 70.1 Ti for the Beckman100-ml Erlenmeyer flask37◦C incubatorUltracentrifuge Beckman L8-70, Optima XL-100 or 1201-ml Mono-Q column (or another with similar characteristics) connected to a

FPLC or AKTA system

Additional reagents and equipment for preparing and homogenizing the brains(Basic Protocol 1), FPLC (Pazzagli and Avila, 1994), and SDS-PAGE (UNIT 6.1)

1. Follow steps 1 to 17 of Basic Protocol 1.

2. Resuspend the pelleted protein in buffer B to a final concentration of 4 mg/ml.Transfer to 10.4-ml centrifuge tubes. Let stand 30 min in an ice-water bath.

3. Centrifuge 15 min at 20,000 × g, 4◦C.

4. Collect the supernatant in a 100-ml Erlenmeyer flask and add GTP, paclitaxel, andpoly-L-aspartic acid to final concentrations of 1 mM, 10 µM, and 50 µM, respectively.Incubate for 30 min at 37◦C.

5. Ultracentrifuge at 30 min at 100,000 × g, 25◦ to 30◦C.

Prewarm the rotor.

mol.mass(kDa)

200

11696

68

45

29

Figure 3.29.5 Characterization of the isolated MAP1A protein (BasicProtocol 4) by denaturing SDS-PAGE (7% polyacrylamide). The gelwas stained with Coomassie blue as explained in UNIT 6.6.


MicrotubuleProteins

3.29.12


6. Load the MAP1-containing supernatant (at a concentration of 0.5 mg/ml) onto a1-ml mono-Q column and fractionate by fast protein liquid chromatography (FPLC;Pazzagli and Avila, 1994). Wash and equilibrate the column with buffer C until theA280 of the eluent returns to the baseline.

7. Next, elute using a linear gradient from buffer C to buffer D.

8. Characterize the protein content of the different collected fractions by SDS-PAGE(UNIT 6.1).

9. Select only MAP1-containing fractions (Fig. 3.29.5) and dialyze against buffer A.Store samples at −20◦C or −70◦C.

Be aware that usually MAP1 proteins are eluted in more than one peak.

BASICPROTOCOL 5

ISOLATION OF HIGH-MOLECULAR-WEIGHT MAP2

The MAP2 gene gives rise to different isoforms that are developmentally regulated. Forinstance, those showing a higher molecular weight are the major isoforms in adult brain(Matus, 1988). As already mentioned, MAP2 is heat resistant and remains stable andfully functional after a high temperature treatment. Consequently, the inclusion of a heat-treatment step in the purification protocol facilitates the separation of MAP2 from othermicrotubule-associated proteins that should denature and precipitate (Herzog and Weber,1978; Sloboda and Rosembaum, 1982). An alternative protocol for MAP2 purificationhas also been published (Williams and Lee, 1982).

Materials

Porcine or bovine brains or alternatively microtubule protein stored at −80◦C orliquid nitrogen (Basic Protocol 1)

Buffer A (see recipe)NaCl2-mercaptoethanolAmmonium sulfateSepharose 4B resin (GE Healthcare-Pharmacia)

Teflon-glass homogenizerIce-water bathCentrifugeCentrifuge rotors: GSA or JA-10, Type 50.2 Ti and 70.1 Ti for the BeckmanBoiling water bath

Additional reagents and equipment for SDS-PAGE (UNIT 6.1)

Isolate MAP21. Follow steps 1 to 17 of Basic Protocol 1.

2. Depolymerize microtubules in cold buffer A using a Teflon-glass homogenizer toobtain microtubule proteins (10 to 20 mg/ml).


3. Incubate in an ice-water bath for 30 min.

4. Centrifuge the mixture 15 min at 25,000 × g, 4◦C.

5. Collect the supernatant and dilute to a protein concentration of 1 mg/ml with bufferA. Then, add NaCl and 2-mercaptoethanol to a final concentration of 1 M and 10 mM,respectively.

6. Heat the resulting protein solution for 5 min at 100◦C in a boiling water bath.


3.29.13



A white precipitate containing denatured protein should be obtained.

8. Then, collect the supernatant in an Erlenmeyer flask and add ammonium sulfate saltto 35% (w/v) saturation. Incubate 10 min at room temperature.

A total of 20.9 g of ammonium sulfate should be added to 100 ml of solution to obtain a35% saturated solution.

9. Centrifuge 10 min at 10,000 × g, 4◦C, then discard the supernatant and resuspendthe pellet in 2 to 5 ml of buffer A containing 1 M NaCl.

10. Load resuspended protein onto a Sepharose 4B column equilibrated with buffer Acontaining 1 M NaCl.

11. Characterize the proteins present in the different collected fractions by SDS-PAGE(UNIT 6.1; Fig. 3.29.6).

Figure 3.29.6 (A) Fractionation of heat-resistant MAPs by chromatography on Sepharose 4B(Basic Protocol 5). (B) SDS-PAGE analysis of the corresponding eluted fractions. The gel wasstained with Coomassie blue as explained in UNIT 6.6. Abbreviations: Vo, void volume.


MicrotubuleProteins

3.29.14


mol.mass(kDa)

200

11696

68

45

29

Figure 3.29.7 Characterization of the isolated high-molecular-weightMAP2 protein (Basic Protocol 5) by denaturing SDS-PAGE (7% acry-lamide). The gel was stained with Coomassie blue as explained inUNIT 6.6.

12. Pool fractions containing MAP2 and precipitate protein by the addition of ammoniumsulfate to 50% saturation. Collect the precipitated protein by centrifuging 10 min at15,000 × g, room temperature.

A total of 31.3 g of ammonium sulfate should be added to 100 ml to obtain a 50% saturatedsolution.

13. Add 1 ml of buffer A to resuspend the pellet and dialyze against 1 liter of the samebuffer.

14. Store protein samples in a freezer at −20◦C (Fig. 3.29.7).

BASICPROTOCOL 6

ISOLATION OF TAU PROTEIN

The primary gene transcript of tau is a well-known example of alternative splicing inthe mammalian brain (Himmler et al., 1989). Furthermore, tau possesses multiple phos-phorylation sites that are selectively used to create polypeptides with different functions.The post-translational modifications of the various tau isoforms result in multiple proteinbehaviors (Lindway and Cole, 1984). For example, not all the phosphorylated tau iso-forms bind to microtubules. For this reason there are two alternate procedures dependingon whether we want to isolate all the isoforms, or just those that bind to microtubules(Garcia de Ancos et al., 1993).

To isolate total tau isoforms (Garcia de Ancos et al., 1993), adjust the concentration ofNaCl and DTT to 1 M and 1 mM, respectively in the homogenate obtained in step 12of Basic Protocol 1, and boil for 5 min and chill on ice. Further steps of the purificationare indicated below. In this protocol we describe the isolation of microtubule binding tauisoforms. In addition to this protocol, there are other tau purification procedures that havealso been described (Herzog and Weber, 1978; Sobue et al., 1981; Drubin and Kirschner,1986; Garcia de Ancos et al., 1993).


3.29.15


Materials

Porcine brainBuffer A (see recipe)Perchloric acidAmmonium sulfateGlycerol

Ice-water bathBoling water bathCentrifugeCentrifuge rotors: GSA or JA-10. Type 50.2Ti and 70.1 Ti for the Beckman

Isolate tau1. Follow steps 1 to 17 of Basic Protocol 1.

2. Resuspend microtubule proteins in 25 ml of buffer A. Leave 30 min in an ice-waterbath.


4. Collect the supernatant in a 100-ml Erlenmeyer flask and correct the concentrationsof NaCl and DTT to 1 M and 1 mM, respectively. Then boil samples for 5 min andimmediately chill on ice for 5 min.

5. Centrifuge samples 30 min at 35,000 × g, 4◦C.

6. Add perchloric acid to the supernatant to a 2.5% (v/v) final concentration. Keep onice for 10 min.


8. Recover the supernatant, collecting it in a 100-ml Erlenmeyer flask, and add ammo-nium sulfate up to 50% saturation. Incubate 30 min at 4◦C.

A total of 313 g of ammonium sulfate should be added to 1 liter to obtain a 50% saturatedsolution.

mol.mass(kDa)

116

96

68

45

29

Figure 3.29.8 Characterization of different isoforms of the isolatedtau protein by denaturing SDS-PAGE (10% acrylamide). The gel hasbeen stained with Coomassie blue as explained in UNIT 6.6.


MicrotubuleProteins

3.29.16



10. Dissolve the pellet in buffer A (to a final concentration of 1 to 5 mg/ml), bring up to2.5% perchloric acid, and centrifuge 15 min at 35,000 × g, 4◦C.

11. Decant supernatant and add glycerol to a final concentration of 25% (v/v). Centrifuge15 min at 35,000 × g, 4◦C.

12. Resuspend the pellet—which should appear as a translucent film stuck to the cen-trifuge tube wall—in buffer A to a final concentration of 0.5 to 2 mg/ml. Storesamples in a freezer at −20◦C (Fig. 3.29.8).

SUPPORTPROTOCOL 1

NONDENATURING POLYACRYLAMIDE GEL ELECTROPHORESIS AS ASIMPLE METHOD TO QUANTIFY/CHECK POLYMERIZATION-COMPETENT TUBULIN

The determination of the purity of the different protein fractions from the various pu-rification steps can be monitorized using SDS-denaturing gels (UNIT 6.1). Purified tubu-lin is usually analyzed by SDS-PAGE. Under denaturing conditions, α- and β-tubulinpolypeptides have different mobilities, both polypeptides in the molecular mass rangeof 55 kDa. However, the analysis and quantification of the amount of tubulin by mostof the known biochemical methods does not provide information about the native stateof the tubulin in the preparation. To distinguish between folded and unfolded tubulin(noncompetent for microtubule assembly) we can take advantage of an old, but stillvery important, nondenaturing gel electrophoresis technique first applied by Ornstein(1964) for the analysis of water-soluble serum proteins (see UNIT 6.5). This method can beused only under conditions that preserve the native state of soluble individual proteinsor complexes. In contrast to classic SDS-denaturing electrophoresis, the migration ofproteins in native gels is essentially dependent on their isoelectric point and the pH ofthe buffer (Safer, 1994). Consequently, in native electrophoresis it is crucial to select theappropriate buffer for each protein under analysis. The behavior of native tubulin undernondenaturing conditions was established by Zabala and Cowan (1992). Although manytimes it is advantageous to use a discontinuous system to maximize the resolution of theprotein bands, we have found that this is not required for tubulin, and it is easier andfaster to prepare a continuous system. To prepare and to run a native electrophoresis,carefully follow the safety indications explained in Chapter 6.

Materials

Gel solutions (Table 3.29.1)4× loading buffer [50 mM MES, pH 6.7, containing 30%(v/v) glycerol or

25%(w/v) sucroseElectrophoresis (running) buffer (see recipe)GTPProtein sample to be analyzed10× transfer buffer (see recipe), optionalElectrophoresis equipment (Chapter 6; e.g., Miniprotean from Bio-Rad and

0.75-mm spacers)

Additional reagents and equipment for gel staining (UNIT 6.2)

1. Assemble the gel glass-plate sandwich and fit it to the gel electrophoresis unit castingstand according to manufacturer’s instructions.

2. Prepare the gel solution as indicated in Table 3.29.1.

3. Pour the gel mix to the top of the gel mold and insert the comb (0.75-mm spacersand 10 wells per comb). Avoid trapping air bubbles under the comb teeth. Allow gelto polymerize for 30 to 60 min.


3.29.17


Table 3.29.1 Recipe for Gel Preparation Using a Native Continuous BufferSystema

Stock solution (ml)b Final acrylamide concentration in gel (%)c

4 5 6 7

Distilled water 6.48 6.15 5.81 5.48

30% acrylamide/0.8%bisacrylamided

1.33 1.66 2 2.33

0.5 M MES, pH 6.7 2 2 2 2

1 M MgCl2 0.01 0.01 0.01 0.01

0.5 M EGTA 0.02 0.02 0.02 0.02

1 M GTP 0.01 0.01 0.01 0.01

10% (w/v) APS(ammonium persulfate)e

0.14 0.14 0.14 0.14

TEMEDf 0.01 0.01 0.01 0.01aPreparation of the gel: Use a 75 or 100-ml Erlenmeyer flask or in order to speed polymerization, degasthe mix under vacuum several minutes in a side-arm flask. Mix the ProtoGelc solution with 0.5M MESpH 6.7, 1M MgCl2, 1M GTP and 0.5M EGTA. Add 10% (w/v) ammonium persulfate and TEMED.bAll reagents and solutions used must be prepared with Milli-Q purified water or equivalent.cThe recipe is for 10 ml solution, which is adequate for 2 minigels (7 cm × 8 cm) 0.75 mm thickness.dThe polyacrylamide can be prepared as described in Chapter 6, but it is more convenient to use aready-to-use commercial solution, we usually use ProtoGel 30% (w/v) acrylamide:0.8% (w/v) bis-acrylamide from National Diagnostics, England.eIt can be prepared and stored frozen at −20◦C in aliquots but we prefer to use freshly made.fAdd just before polymerization.

4. Mix the protein sample to be analyzed with 4× native loading buffer [50 mM MES,pH 6.7, containing 30% (v/v) glycerol or 25% (w/v) sucrose] for a final concentrationof 1×.

For better sample visualization small amounts [0.025% (w/v)] of bromphenol blue canbe added in the loading buffer.

5. Assemble the gel unit and fill the upper tank with enough electrophoresis buffer withGTP added to 0.01 M. Use the rest of the 800 ml of electrophoresis buffer, withoutGTP, to fill the rest of the cell. Remove the comb and rinse wells with buffer.

6. Carefully load a maximum of 25 µl of the samples starting up from the bottom ofthe wells.

Use a micropipet or alternatively a Hamilton syringe to load the gel.

7. Connect the unit to the power supply. Set at constant voltage of 80 V for 2 hr.

We usually run one gel per cell unit, for no longer than 2 hr at room temperature.

Gels can run longer than 2 hr if the electrophoresis buffer is replaced with freshly preparedelectrophoresis buffer in order to maintain the pH. The apparatus needs to be turned offbefore exchanging the buffer.

8. Turn off the power supply. Disassemble the unit and remove gel from glass sandwich.Stain the gel according to UNIT 6.6.

Coomassie blue or silver staining is the usual method to detect tubulin depending on theamount of tubulin loaded into the gel (Fig. 3.29.9). Alternatively, gels can be transferredonto nitrocellulose or PVDF membranes prior to antibody detection in immunoblots(UNIT 6.2) by using a typical transfer buffer where methanol is omitted.


MicrotubuleProteins

3.29.18


1 2 3 4 5 6 7 8

A

B

at room temp. at room temp.

0 2 hr 4 hr 5 min 0 2 hr 4 hr 18 hr

-dimers

α-tubulin

β-tubulin

50°C

Figure 3.29.9 Assessment of the quality of native tubulin heterodimers by non-denaturing anddenaturing gel electrophoresis. (A) Nondenaturing PAGE (8.5% acrylamide) characterization of1-µl aliquots (Support Protocol 1) of purified tubulin (at 6 µg/µl) diluted to 10 µl with water Milli-Q(lanes 1 to 4) or 50 mM MES, pH 6.7 (lanes 5 to 8), and incubated for the indicated times atroom temperature with the exception of the aliquot analyzed in lane 5, which was incubated 5 minat 50◦C. (B) The same tubulin samples were loaded on a gel prepared and run as described inSupport Protocol 1. Both gels were stained with Coomassie blue and then scanned before drying.For color version of this figure see http://www.currentprotocols.com.

SUPPORTPROTOCOL 2

VISUALIZATION OF POLYMERIZED MICROTUBULES BY ATOMICFORCE MICROSCOPY (AFM)

Atomic Force Microscopy (AFM) is a powerful technique with a wide range of appli-cations in biology, allowing not only imaging, but also different types of probing andmanipulation at nanometric scale. AFM systems are becoming widely available, and newdevelopments are reported everyday, many of them in biological research. In AFM a sharptip (local probe) is moved over a surface at constant (van der Waals) interaction force be-tween the tip and the surface, allowing the reconstruction of the surface topography. Thismeasurement process can be performed in different ambient conditions, e.g., in vacuum,air, or liquid, over a broad temperature range. These characteristics and the possibility toobserve features at nanometric scale in a nondestructive manner are a strong motivationfor the use of AFM on biological systems. As compared to existing microscopy tech-niques in the same range (as electron microscopy), sample preparation is also much easierand less time-consuming, allowing one to perform measurements even on live systems(Fotiadis et al., 2002). The list of examples is now almost endless. For example, AFM


3.29.19


techniques have been extensively used to image the surface of cells (Dufrene et al., 1999;Charras and Horton, 2002), nucleic acids (Pande et al., 1998; Scheuring et al., 1999),proteins (Round et al., 2002), chromosomes (Radmacher et al., 1994; Fritzsche et al.,1997), viruses (Dubrovin et al., 2007), and biomaterials (Wallwork et al., 2001). Besidesbeing used as a powerful imaging technique, AFM shows a strong potential as a uniquetool for nanometric probing of physical (e.g., elasticity or viscosity of surfaces, bindingforces, strength of protein folding) and chemical properties (by using a chemically ac-tivated tip) and for precise local manipulation (Rief, et al., 1997; Sritharan et al., 1998;Raab et al., 1999; Dufrene, 2000). It is nowadays a fundamental tool for nanomedicine(medicine approached at a molecular level) and its relevance can no longer be overlooked

Microtubules may be characterized by AFM (Ramalho et al., 2007), which permitsnanometer-scale resolutions and topographic characterization of the sample, allowingone to visualize the assembled microtubules.

Here we introduce a protocol adapted from Martins et al. (2005) and Moreno-Herreroet al., (2002, 2005) describing how to adsorb microtubules on a substrate to visualize themby AFM. In this protocol, microtubules are adsorbed to mica functionalized with APTES,a silane with an amine group that is positively charged at around pH 7. Since mica iscomposed of layers, which can be cleaved (“peeled” off) to expose a new clean surfaceand, unlike more traditional glass, no preliminary washing procedures are necessary.

Materials

Mica (muscovite; SPI Supplies)Nail polish or water-resistant glue (e.g., epoxy resin)Silanization solution: 0.1% (v/v) 3-aminopropyl-triethoxysilane (APTES; Fluka,

96%) freshly prepared with Milli-Q waterMilli-Q water; if unavailable, bidistilled water filtered through a 0.22-µm pore filterGTPPaclitaxel (taxol)Sample of purified tubulin (Basic Protocol 3)0.1 M PIPES, pH 6.91 mM EGTA1 mM MgCl21 mM PMSF

Sharp scissorsGlass coverslips or slidesDuct tapeMicropipet30◦C thermostatic bathAFM Si tapping mode tips (Olympus, OTESP)

Functionalize the mica1. Use sharp scissors to cut mica squares of appropriate dimensions (2 to 5 mm) and

mount them on glass coverslips or slides (for easier handling) with nail polish orwater-resistant glue (e.g., epoxy resin). Remove the upper layers of the mica squaresby pulling off the top layer with a piece of duct tape.

2. Immediately apply 10 µl silanization solution on the exposed surface, taking carenot to spill on the sides. To complete silanization let it rest for 10 min.

While the nail polish will secure the mica even if wet, silanization solution may still getbetween the mica layers and distort them.


MicrotubuleProteins

3.29.20


Figure 3.29.10 (A) AFM topography images of polymerized microtubules adsorbed to silanized mica where a fewindividual microtubules with a well preserved structure are easily observed. (B) A microtubule-rich region in the sampleshowing contaminant material. (C) Distortion of measurements due to the contaminants in a dirty sample. For color versionof this figure see http://www.currentprotocols.com.

3. Remove the silanization solution as much as possible with a micropipet and avoidscratching the mica surface. Wash off the mica three times, each time with 10 µl ofMilli-Q water and allow the mica to air dry.

It is quite important that mica surface is maintained clean during this process in order toguarantee that environmental dust does not stick to it during the process of silanization.

Polymerize and adsorb microtubules4. Add 2 µl of 100 mM GTP (to a final concentration of 1 mM) to 200 µl of a purified

tubulin solution in 0.1 M PIPES pH 6.9, 1 mM EGTA, 1 mM MgCl2, 1 mM PMSF,at 1 mg/ml on ice.

5. Transfer to a 30◦C thermostatic bath and incubate for 45 min.

6. Add 4 µl of 1 mM paclitaxel (taxol) to give a final concentration of 20 µM. Incubatefor another 15 min at the same temperature.

Optionally, microtubules may be concentrated by sedimenting them at 15,000 × g for120 min. This will usually require more than one aliquot to give a visible pellet.

If you are not going to immediately use the assembled microtubule suspension then aliquotit in suitable volumes, promptly freeze in liquid nitrogen, and store at −80◦C.

7. When appropriate, thaw the microtubule suspension by placing on ice. Apply 5 µl ofthe microtubule suspension on the surface of the functionalized mica. Incubate for 5to 10 sec. Immediately carefully remove the liquid with a micropipettor.

8. Gently wash the preparation twice with 10 µl Milli-Q water. Allow remaining waterto air dry and observe with AFM as soon as possible.

Avoid touching the mica surface with the tip of the micropipet in steps 1 and 2.

For AFM measurements we use a Dimension 31 00 (Veeco/Digital Instruments) mi-croscope with nanoscope III a controller and silicon nitride tips (SiN2). All sam-ples are observed in tapping mode at atmosphere in order to minimize de-adsorption(Fig. 3.29.10).


3.29.21


REAGENTS AND SOLUTIONSUse deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

BRB80 buffer

80 mM PIPES, pH 6.81 mM EGTA1 mM MgCl2Store up to 2 months at 4◦C

Buffer A, 10×1 M MES, pH 6.720 mM EGTA10 mM MgCl2Store up to 2 months at 4◦C

Buffer B

0.15 M PIPES, pH 7.0 (see recipe)2 mM EGTA0.1 mM MgCl2Store up to 2 months at 4◦C

Buffer C

0.1 M PIPES, pH 7.0 (see recipe)2 mM EGTAStore up to 2 months at 4◦C

Buffer D

0.1 M PIPES, pH 7.0 (see recipe)2 mM EGTA1 M NaClStore up to 2 months at 4◦C

EGTA, 0.5 M

Dissolve 19 g EGTA [ethylene glycol-bis-(2-aminoethyl)-N,N,N′,N′-tetraaceticacid] in 80 ml water. Titrate to pH 8 with KOH or NaOH. Adjust to a final volumeof 100 ml. Store up to 2 years at room temperature.

Electrophoresis (running) buffer

0.1 M MES, pH 6.71 mM MgCl21 mM EGTA

Dilute and adjust with distilled water 160 ml 0.5 M MES, pH 6.7 (see recipe),800 µl 1 M MgCl2, and 1.6 ml 0.5 M EGTA (see recipe) to 800 ml. Prepare freshjust prior to use.

GTP, 1 M

1.5 ml distilled H2O1 g Na2GTPBring to final volume of 1.91 ml with H2O.

Do not let stand at room temperature or at 4◦C. Aliquot in small volumes and store at−80◦C.


MicrotubuleProteins

3.29.22


Isotonic buffer

0.32 M sucrose1 mM EGTA1 mM MgCl210 mM phosphate buffer, pH 7 (APPENDIX 2A)Store up to 2 months at 4◦C

MES, 0.5 M, pH 6.7

Dissolve 53.3 g MES monohydrate in 400 ml water. Titrate to pH 6.7 with KOH orNaOH. Adjust to a final volume of 500 ml with water. Store up to 6 months at 4◦C.

Paclitaxel, 10 mM

10 mM paclitaxel (taxol; Sigma). Prepare in DMSO. Store 50-µl aliquots at −80◦Cand use at final concentration of 20 µM.

PIPES, 1 M (pH 7.0)

151.2 g PIPES (free acid)H2O to 400 mlAdd concentrated KOH dropwise to achieve pH 7.0.Bring to final volume of 500 ml with H2OStore up to 2 months at 4◦C

Protease inhibitors

100 mM phenylmethylsulfonyl fluoride, (PMSF; APPENDIX 2A) prepare fresh, use at1 mM, dissolve in DMSO

1000× leupeptin, prepare 1 µg/µl in Milli-Q water1000× pepstatin A, prepare 1 µg/µl in DMSO1000× aprotinin, 10 µg/ml in Milli-Q water (sodium azide or 0.2% Triton X-100

can be added to avoid contamination)

CAUTION: Phenylmethylsulfonyl fluoride is toxic.

Transfer buffer, 10×Dissolve the following in water:15.13 g Tris base (250 mM)72.067 g glycine (1.92 M)Adjust to a final volume of 500 ml with H2O

COMMENTARY

Background InformationThe generation of complex neuronal mor-

phologies during vertebrate neurodevelop-ment is characterized by the production oflong cytoplasmic processes known as neurites.These processes, referred to as axons and den-drites, will eventually generate a complex net-work of neuronal synaptic contacts. Althoughmicrotubules are universal polymers in all eu-karyotic cell types, in neurons they are highlyspecialized and more abundant, and play cru-cial roles in the determination of the cell shape,being organized in long bundles within the ax-ons and dendrites of mature neurons. Micro-

tubules are dynamic structures in vitro, wherepossible regulatory factors of the process havebeen thoroughly studied. These investigationsled to the discovery of a group of proteins thatbind to tubulin “in vitro” during microtubulepolymerization assays, promoting the poly-merization and further stabilizing the poly-mer structure. These proteins are referred toas microtubule-associated proteins or MAPs.Three major families of MAPs have been iden-tified and characterized: MAP1 (Vallee, 1990),MAP2 (Murphy et al., 1977), and tau proteins(Cleveland et al., 1977).


3.29.23


MAP1 proteinsThe MAP1 protein family consists of two

distinct, but related, polypeptides, MAP1Aand MAP1B (Schoenfeld et al., 1989).MAP1A has a molecular mass of 299 kDa,whereas MAP1B has a molecular mass of255 kDa as predicted from their respectiveamino acid sequences (Noble et al., 1989;Langkopf et al., 1992).

There are also three low-molecular-weightproteins associated with the microtubule-binding domains of both MAP1A andMAP1B; these are referred to as light chains:LC1 (34 kDa), LC2 (30 kDa), and LC3(19 kDa; Schoenfeld et al., 1989).

In the mammalian brain there is a strongdevelopmental control of the expression pat-terns of these MAPs. The first MAP expressedin neurons is MAP1B (Tucker et al., 1988).Immunohistochemical analyses in developingbrain tissue have shown that this protein ishighly concentrated in the growing axons ofthe developing neurons, exhibiting a moremoderate expression in both axons and den-drites of mature neurons (Schoenfeld et al.,1989). Indeed, MAP1B expression is down-regulated during brain development (Binderet al., 1984; Bloom et al., 1985; Riederer et al.,1986; Tucker et al., 1989; Garner et al., 1990).On the contrary, MAP1A is more abundant inmature neurons (Bloom et al., 1984).

MAP2There are several developmentally regu-

lated isoforms of MAP2 arising from alter-native mRNA splicing. MAP2B is a high-molecular-weight isoform which contains1828 amino acids and has an apparent molec-ular mass of 270 kDa as determined by SDS-PAGE (Lewis et al., 1988). Also, MAP2Ashows a slower electrophoretic mobility(Matus, 1988). There is a smaller form, re-ferred to as MAP2C that contains 467 aminoacids and has an apparent molecular mass of70 kDa as determined by SDS-PAGE. MAP2Bis a neuron-specific phosphoprotein which isselectively localized in the cell bodies and den-drites (Caceres et al., 1984; Huber and Ma-tus, 1984). MAP2C is expressed perinatallyin rats, coincident with the period of maxi-mal dendritic outgrowth and synaptogenesis(Riederer and Matus, 1985). There is an addi-tional MAP2 isoform which is more abundantin glial cells named MAP2D (Doll et al., 1993).

TauThere are several tau proteins with appar-

ent molecular masses ranging from 55 kDa

to 68 kDa (as determined by SDS-PAGE);they are found in the central nervous system(Weingarten et al., 1975). This large number oftau isoforms is generated by alternative splic-ing from a primary transcript (Himmler et al.,1989; Goedert et al., 1992). Based on theirdomain characteristics, two classes of tau iso-forms have been described. One class containsthree tubulin-binding motifs that are predom-inantly expressed in the developing brain; asecond class contains four motifs and is mostlyexpressed in the adult brain (Kosik et al., 1989;Goedert et al., 1991).

An additional tau isoform, with an appar-ent molecular mass of 110 kDa, has also beenidentified in the peripheral nervous system(Georgieff et al., 1991). This high-molecular-weight tau contains four repeated motifs inits microtubule-binding domain; it is similarto the adult brain tau isoform but has an ad-ditional insertion of 254 amino acids in itsamino-terminal region (Goedert et al., 1991;Couchie et al., 1992).

Critical Parameters andTroubleshooting

Our experience suggests that, although ahigher yield of microtubule proteins is usuallyobtained from porcine brain tissue, cow brainsdo also give good tubulin yields. Tubulin hastraditionally been considered an extremely la-bile protein that becomes noncompetent forpolymerization if left on ice for long periods oftime. However, as Figure 3.29.9 shows, tubulinis a stable dimer for >4 hr at room tempera-ture in MES buffer at pH 6.7 (at a concen-tration as low as 0.6 µg/µl, with no magne-sium ions or GTP added). In fact, the tubu-lin dimer becomes unstable only after 18 hror longer incubation periods. Our experiencealso shows that tubulin can be stored at tem-peratures between −70 and −80◦C. Regardingthe size of the preparation, we usually pre-pare medium-scale tubulin preparations. Thisguarantees a faster protein extract prepara-tion, which is key to preserving a good-qualitytubulin, as explained in the Time Consider-ations section. The intermediate-step tubulinaliquots obtained can be fast-frozen and storedat −80◦C instead of in liquid nitrogen. Foreach experiment, appropriate-size aliquots canbe thawed, mixed well by flicking, and cen-trifuged 20 min at 20,000 × g, 4◦C, beforeuse, to discard insoluble material. Although re-peated freezing and thawing of aliquots is notrecommended, we find that tubulin will stillwork fine in freeze-thawed aliquots. Another


MicrotubuleProteins

3.29.24


crucial point to obtaining a good homogenatewhen isolating tubulin from brain extracts isnot to destroy the tissue excessively. The useof a classical blender, even at low speed, is notrecommended since vigorous homogenizationleads to the formation of aggregates that persistduring the purification. Moreover, this proce-dure also provokes the formation of tubulinaggregates. We recommend the use of a Poly-tron set at low speed, or just a Teflon-in-glasshomogenizer at 2,000 rpm for 5 to 10 sec,with the pestle moving up and down the ho-mogenizer. A commercial drill adapted as ahomogenizer is a cheaper alternative.

Preparation of microtubule samples forAFM entails adsorption onto an adequate sub-strate (positively charged) and washing off thebuffer as much as possible in order to avoid ar-tifacts resulting from salt crystallization. Oth-erwise, salt crystals will rapidly grow overthe mica surface after the microtubule suspen-sion has dried, completely impairing the AFMmeasurements. Washes must be carefullyperformed to avoid microtubule damage orremoval from the surface. So, drop and re-move water from the surfaces containing mi-crotubules during washes.

Since microtubules are adsorbed to micafunctionalized with APTES, the time of incu-bation of microtubules on the functionalizedmica is one of the most important parametersin this method. Longer incubation times tendto yield “dirty” samples due to the adsorptiononto the surface of nonpolymerized tubulinand other contaminants (even if they are scarcein the sample). On the other hand, shorter incu-bation times will result in sparser distributionsof microtubules on the surface, allowing a bet-ter visualization. The more microtubules areadsorbed onto the surface, the more difficultit will be to observe detailed features. Incu-bation times of 5 to 10 sec will usually givegood results with microtubules polymerizedfrom ∼1 mg/ml brain tubulin solution. How-ever, longer times may be necessary for lowerconcentrations. Attention should also be paidto the concentration of microtubules in sus-pension, since an increase in polymer concen-tration would lead to aggregation and micro-tubule bundle formation.

Anticipated ResultsThe cytosolic extract obtained after the first

centrifugation of the isolation procedure of mi-crotubule proteins usually contains a proteinconcentration in the range of 20 to 40 mg/mlof protein and ∼4 to 8 mg/ml of tubulin.Phosphocellulose-FPLC chromatography pu-

rified bovine brain tubulin should give a sin-gle band upon electrophoresis under nativeconditions—in the presence of GTP—whichserves as a reference marker for native tubu-lin heterodimers. The amount of tubulin in thisunique band is extremely useful to quantify theamount of tubulin dimers capable of polymer-ization. Proteins that have denatured or ag-gregated during these purification steps willremain at, or close to, the origin of the nativegel (Fig 3.29.9). Therefore, this type of elec-trophoresis will serve to estimate the amountof tubulin that should be used in any other ex-periment requiring native tubulin. The use ofGTP during native gel electrophoresis is crit-ical for this type of analysis. In the absenceof GTP we have found that the intensity ofthe native tubulin heterodimer band decreases,indicating that tubulin dimers have became un-stable in the absence of GTP. This could be dueto denaturation of the β-tubulin that loses GTPduring the running of native gel electrophore-sis in the absence of this nucleotide (Zabalaand Cowan, 1992). Be aware that ATP (usedinstead of GTP for actin) cannot replace GTPin this type of electrophoresis.

There are a number of different gel stain-ing protocols that can be used (UNIT 6.6).Coomassie blue or silver staining are theusual ways to detect tubulin depending on theamount of sample loaded on the gel. Protein inthese gels can also be transferred to nitrocel-lulose or PVDF membranes prior to antibodydetection by immunoblots (UNIT 6.2). In thiscase, a typical transfer buffer without methanolshould be used.

In vitro polymerized microtubule samplesobserved by AFM are typically heterogeneous.These samples will characteristically presentareas with fewer microtubules, but also fewerinterfering contaminants (Fig. 3.29.10A),along with areas denser in microtubules butpresenting more contaminants (Fig. 3.29.10B)or areas with many amorphous structures(precipitates from the microtubule solution)that will make the analysis more difficult(Fig.3.29.10C). Denser areas, as well as bun-dles of microtubules, and generally more con-taminants, are often observed in samples pre-pared from more concentrated microtubulesolutions. On the other hand, concentrationslower than those recommended will result invery scattered samples where microtubulesmay be difficult to find.

Time ConsiderationsThe most critical step in the tubulin protein

purification protocol is the time spent from the


3.29.25


time of slaughter to the end of the first micro-tubule polymerization. It is crucial to obtainbrains of freshly sacrificed animals and to re-move the skulls as soon as possible. Thesemust be immediately stored at 4◦C wrappedin plastic bags or similar wrapping material,to avoid direct contact with ice. The brain cy-tosolic protein extract should be prepared assoon as possible. Everything needed for theisolation/purification procedure should be al-ready prepared in the laboratory. In the sameway, extreme care should be taken to avoid anydelay in washing and cleaning the brains.

For AFM, waiting periods for sample dry-ing should typically be of 0.5 to 1 hr, depend-ing on room temperature and humidity. Forreproducibility, microtubule incubation timesmust be carefully observed, even if chosentimes differ from those detailed in this pro-tocol. Nonetheless, this procedure should takea maximum of 4 hr. Similarly, samples mustbe observed with AFM as soon as micro-tubules are adsorbed to functionalized mica,thus avoiding artifacts caused by dust or degra-dation. Samples can be kept for at least a weekat 4◦C (in a sealed container) although somedegradation and contamination can occur.

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Internet ResourcesWeb sites for protocols and advice on microtubule

preparation, tubulin purification, and handling:

http://mitchison.med.harvard.edu/protocols/tubprep.html

Web site for large scale tubulin preparation proto-col.

http://mitchison.med.harvard.edu/protocols/recycle.html

http://www.bio.com/protocolstools/protocol.jhtml?id = p1426

http://www.ciwemb.edu/labs/koshland/Protocols/MICROTUBULE/cyclingmicrotub.html

Web sites for handling recycled tubulin.


Protocol for tubulin/MAP co-sedimentation underhigh ATP conditions and MAP/motor/tubulin co-sedimentation.


MicrotubuleProteins

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In this protocol purified tubulin is stabilized withTaxol.

http://www.bio.unc.edu/faculty/salmon/lab/protocolsporcinetubulin.html

Web site for tubulin purification from 3 pig brainssimilar to Basic Protocol 1.

http://www.borisylab.northwestern.edu/pages/protocols.html

Web page for microtubule protein preparation.

isolation of microtubules and microtubule proteinsbiochem.du.ac.in/web/uploads/10 isolation...

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